Liver damage occurs in a number of acute and chronic clinical conditions, including drug-induced hepatotoxicity, viral infections, vascular injury, autoimmune disease and blunt trauma. In addition, patients subject to inborn errors of metabolism may be at risk for developing liver damage. Symptoms of liver damage occurring as a result of these clinical conditions include, for example, fulminant hepatic failure with cholestasis, hepatic lesions, and liver tissue necrosis, and in many instances, the restoration of normal liver function is vital to the survival of patients.
Hepatotoxic compounds can induce almost all types liver injury (Benhamou, J-Pierre, Liver Cells and Drugs, Chapter 164, pgs. 3-12, Colloque INSERM/John Libbey Eurotext Ltd., edited by A. Guillozo (1988). The susceptibility of the liver to damage by chemical agents may be related to its primary role in drug metabolism or is a consequence of hypersensitivity reactions. Up to 25% of cases of fulminant hepatic failure may be the result of adverse reactions to medical agents. Hepatotoxic compounds are also an important cause of chronic liver disease including fatty liver, hepatitis, cirrhosis and vascular and neoplastic lesions of the liver. (Sinclair et al., Textbook of Internal Medicine, 569-575 (1992) (editor, Kelley; Publisher, J. B. Lippincott Co.).
Hepatotoxic compounds may induce liver damage by cytotoxicity to the liver directly or through the production of toxic metabolites (this category includes the hypersensitivity reaction which mimics a drug allergy); cholestasis, an arrest in the flow of bile due to obstruction of the bile ducts; and vascular lesions, such as in veno occlusive disease (VOD), where injury to the vascular endothelium results in hepatic vein thrombosis. Individual susceptibility to liver damage induced by hepatotoxic compounds is influenced by genetic factors, age, sex, nutritional status, exposure to other drugs, and systemic diseases (Sinclair et al., Textbook of Internal Medicine, Supra). Hepatotoxic compounds known to induce liver damage include acetaminophen, nitrosoureas, used in the treatment of cancer, and isoniazid, used in the treatment of tuberculosis.
Although in minor liver damage induced by hepatotoxic compounds, withdrawal of the causative agent may be sufficient to substantially reverse the damage occurred, in many instances where fulminant hepatic failure ensues, aggressive medical therapy, including the administration of antidotes, such as N-acetylcysteine, may be required. A antidotal treatment is, however, often not effective when given more than about 10-24 hours after exposure to the hepatotoxic compound (Goodman and Gilman's. The Pharmacological Basis of Therapeutics 8th edition, Gilman et al., Pergamon Press, 658-659 (1990)). If this happens, the liver damage may become permanent and life threatening, leaving liver transplantation as the only remedy.
Radiation therapy can also induce liver damage. It has been shown that hypoalbuminemia and decreased hepatic blood flow, both symptoms of liver damage, occur after single-dose total body irradiation (Moulder, J. et al. Int J Radiat Oncol Biol Phys 19: 1389-1396 (1990)). Awwad, H. et al., Int J Radiat Oncol Biol Phys 19(5): 1229-1232 (1990) show that lung and hepatic toxicities constitute the main radiation-related damage after half-body irradiation used as the treatment for patients with non-Hodgkin's lymphomas and recommend low dose-rate or multifraction irradiation in order to reduce the risk of liver toxicity. McCracken, J. et al., Cancer Treat Rep 69(1): 129-31 (1985) caution that combined radiotherapy and intra-arterial chemotherapy may result in significant chronic liver damage, as monitored by serum enzyme levels, and recommend exercising caution in the future use of the therapy. Fajardo, L. et al. Arch Pathol Lab Med 104(11): 584-8 (1980) show that radiation-induced liver disease is characterized structurally by progressive fibrous obliteration of central veins (VOD) and that in several patients, VOD occurred at radiation doses conventionally considered safe.
Inborn errors of metabolism exist which result in liver damage. Patients who have a genetically limited capacity to convert aryl epoxides to nontoxic dihydriols, seem predisposed to developing liver damage from exposure to phenytoin and halotane, drugs useful as anesthetics. Also, susceptibility to contraceptive steroid-associated cholestasis appears to have a strong genetic component (Sinclair et al., Textbook of Internal Medicine, Supra).
Liver damage of any origin can be diagnosed and monitored by biochemical tests of liver markers, such as assessment of hepatic blood flow or prothrombin clotting time, or serum markers, such as serum bilirubin, serum transaminase, and serum alkaline phosphatase levels and (Cornelius, C., Hepatotoxicology pg, 181, (1991) and (Awwad, H. Int J Radiat Oncol Biol Phys 19(5): 1229-1232 1990)). Liver damage can also be monitored from histological evaluation of liver tissue, which is helpful in determining the type and extent of liver damage (Sinclair, S. Textbook of Internal Medicine, Supra. It is known that results from in vitro biochemical tests measuring liver function or serum markers and/or results from liver tissue biopsy, correlate with in vivo liver damage assessment. Often, a combination of biochemical tests, tissue biopsy, patient medical history, and assessment of means inducing liver damage is used in determining the extent of liver damage.
Liver cell (hepatocyte) regeneration is believed to be controlled by various growth stimulatory and growth inhibitory cytokines of autocrine or paracrine origin, however, the exact role and action mechanism of these factors is far from entirely understood.
In vitro, DNA synthesis in isolated hepatocytes has been shown to be stimulated by growth factors such as epidermal growth factor (EGF) and type a transforming growth factor (TGF-.alpha.) and to be inhibited by interleukin 1.beta. (IL-1.beta.) (Nakamura et al., Exp. Cell Res., 179: 488-497 (1988)), transforming growth factor .beta.1 (TGF-.beta.1) (Braun et al., Proc. Natl. Acad. Sci. USA, 85: 1539-1543 (1988); Nakamura et al., Biochem. Biophys. Res. Comm., 133: 1042-1050 (1985); Carr et al., Cancer Res., 46: 2330-2334 (1986); Castilla et al., New Eng. J. Med., 324: 933-940 (1992); Houck et al., J. Cell. Physiol., 135: 551-555 [1988]; Strain et al., Biochem. Biophys. Res. Commun., 145: 436-442 (1987)), and activin (U.S. patent Application Ser. No. 07/712,284 filed 10 Jun. 1991). TGF-.beta.1 has been shown to inhibit in vivo DNA synthesis taking place after partial hepatectomy. Russell et al., Proc. Natl. Acad. Sci. USA, 85: 5126-5130 (1988). Vascular endothelial growth factor (VEGF), an endothelial cell mitogen, is expressed in the normal liver (Berse, et al., Mol. Biol. Cell, 3(2): 211-220 (1992)), where it plays a role in tissue nutrition and waste removal.
More recently, a further protein, named hepatocyte growth factor (HGF) has been shown to be a complete mitogen for primary hepatocytes. Although based upon the observation that the level of HGF in the serum rapidly increases following experimental damage to the liver and in patients with fulminate hepatic failure it has been proposed that HGF may be an important mediator of liver regeneration in vivo, and certain experimental evidence supports this hypothesis, there is no clear consensus among scientists about the role of HGH in liver regeneration. Rosen et al., Cell Growth and Differentiation 2: 603 (1991) caution that markedly elevated HGF levels in patients with chronic liver disease may indicate that HGF is a marker for or instigator of human liver damage rather than a repair factor.
Growth factors, proteins with growth factor-like activities, such as cytokines, (Andus et al., Hepatology 13(2): 364-375 (1991)) and therapeutics, such as tissue plasminogen activator (Baglin, et al., Bone Marrow Transplant 5(6): 439-441 (1990)), have been indicated in the treatment of liver damage.
HGF was purified by Nakamura et al. from the serum of partially hepatectomized rats (Biochem. Biophys. Res. Comm. 122: 1450-1459 (1984)). Subsequently, HGF was purified from rat platelets, and its subunit structure was determined (Nakamura et al., Proc. Natl. Acad. Sci. USA, 83, 6489-6493 (1986); and Nakamura et al., FEBS Letters 224, 311-316 (1987)). The purification of human HGF (hHGF) from human plasma was first described by Gohda et al., J. Clin. Invest. 81, 414-419 (1988). According to the results reported by Gohda et al. hHGF is more effective in the stimulation of cultured hepatocyte proliferation than human epidermal growth factor (hEGF) or insulin, and the effect of hHGF with the maximal effects of hEGF and insulin is "additive or synergistic". Similarly, Zarnegar et al., Cancer Research 49, 3314-3320 (1989) described the purification of a polypeptide growth factor, called human hepatopoietin A (HPTA) having very similar properties to hHGF as characterized in earlier publications. As the authors do not disclose the amino acid sequences of their purified proteins, the degree of the structural similarity between the two factors can not be determined.
The N-terminal amino acid sequence of rabbit HPTA was described by Zarnegar et al., Biochem. Biophys. Res. Comm. 163, 1370-1376 (1989).
Both rat HGF and hHGF have been molecularly cloned, including the cloning and sequencing of a naturally occurring variant lacking 5 amino acids in the Kringle 1 (K1) domain, designated "delta5 HGF" (Miyazawa et al., Biochem. Biophys. Res. Comm. 163: 967-973 (1989); Nakamura et al., Nature 342: 440-443 (1989); Seki et al., Biochem. and Biophys. Res. Commun. 172: 321-327 (1990); Tashiro et al., Proc. Natl. Acad. Sci. USA 87: 3200-3204 (1990); Okajima et al., Eur. J. Biochem. 193: 375-381 (1990)). The sequences reported by Miyazawa et al. and Nakamura et al. for hGH differ at several positions. The comparison of the amino acid sequence of rat HGF with that of hHGF revealed that the two sequences are highly conserved and have the same characteristic structural features. The length of the four Kringle domains in rat HGF is exactly the same as in huHGF. Furthermore, the cysteine residues are located in exactly the same positions; an indication of similar three-dimensional structures (Okajima et al., Supra; Tashiro et al., Supra).
A naturally occurring hHGF variant has recently been identified which corresponds to an alternative spliced form of the hHGF transcript containing the coding sequences for the N-terminal finger and first two kringle domains of mature hHGF (Chan et al., Science 254: 1382-1385 (1991); Miyazawa et al., Eur. J. Biochem. 197: 15-22 (1991)). This variant, designated HGF/NK2, has been proposed to be a competitive antagonist of mature hHGF.
The HGF receptor has been identified as the product of the c-Met proto-oncogene (Bottaro et al., Science 251: 802-804 (1991); Naldini et al., Oncogene 6: 501-504 (1991)), and 190-kDa heterodimeric (a disulfide-linked 50-kDa a-chain and a 145-kDa .beta.-chain) membrane-spanning tyrosine kinase protein (Park et al., Proc. Natl. Acad. Sci. USA 84: 6379-6383 (1987)). The c-Met protein becomes phosphorylated on tyrosine residues of the 145-kDa .beta.-subunit upon HGF binding.
The levels of HGF increase in the plasma of patients with hepatic failure (Gohda et al., Supra) and in the plasma (Lindroos et al., Hepatol. 13: 734-750 (1991)) or serum (Asami et al., J. Biochem. 109: 8-13 (1991)) of animals with experimentally induced liver damage. The kinetics of this response is rapid, and precedes the first round of DNA synthesis during liver regeneration suggesting that HGF may play a key role in initiating this process. Although HGH was originally thought to be a liver-specific mitogen, more recently, it has been shown to be a mitogen for a variety of cell types including melanocytes, renal tubular cells, keratinocytes, certain endothelial cells and cells of epithelial origin (Matsumoto et al., Biochem. Biophys. Res. Commun. 176: 45-51 (1991); Igawa et al., Biochem. Biophys. Res. Commun. 174, 831-838 (1991); Han et al., Biochem. 30: 9768-9780 (1991); Rubin et al., Proc. Natl. Acad. Sci. USA 88: 415-419 (1991)). Interestingly, HGF can also act as a "scatter factor", an activity that promotes the disassociation of epithelial and vascular endothelial cells in vitro (Stoker et al., Nature 327: 239-242 (1987); Weidner et al., J. Cell Biol. 111: 2097-2108 (1990); Naldini et al., EMBO J. 10: 2867-2878 (1991)). Moreover, HGF has recently been described as an epithelial morphogen (Montesano et al., Cell 67: 901-908 (1991)). Therefore, HGF has been postulated to be important in tumor invasion and in embryonic development. Chronic c-Met/HGF receptor activation has been observed in certain malignancies (Cooper et al., EMBO J. 5: 2623 (1986); Giordano et al., Nature 339: 155 (1989)).
Activin consists of a homodimer or heterodimer of inhibin .beta. subunits, which may be .beta..sub.A or .beta..sub.B subunits. Vale et al., Recent Prog. Horm. Res., 44: 1-34 (1988). There is 95-100% amino acid conservation of .beta. subunits among human, porcine, bovine, and rat activins. The .beta..sub.A and .beta..sub.B subunits within a given species are about 64-70% homologous.
The activin .beta..sub.A and .beta..sub.B homodimers ("Activin A" and "Activin B," respectively) have been identified in follicular fluid, and both molecules have been cloned and their genes expressed. Mason et al., Biochem. Biophys. Res. Commun., 135: 957 (1986); EP Pub. No. 222,491 published May 20, 1987; Mason et al., Molecular Endocrinol., 3: 1352-1358 (1989); Schwall et al., Mol. Endocrinol., 2: 1237-1242 (1988); Nakamura et al., J. Biol. Chem., 267: 16385-16389 (1992). The complete sequence of the .beta..sub.B subunit is published in Serono Symposium Publications, entitled "Inhibin-Non-Steroidal Regulation of Follicle Stimulating Hormone Secretion", eds. H. G. Burger et al., abstract by A. J. Mason et al., vol. 42, pp. 77-88 (Raven Press, 1987), entitled "Human Inhibin and Activin: Structure and Recombinant Expression in Mammalian Cells." The recombinant molecule has been shown to increase serum levels of FSH in rats when delivered by subcutaneous injection. Schwall et al., Endocrinol., 125: 1420-1423 (1989); Rivier and Vale, Endocrinol., 129: 2463-2465 (1991).
Activin was initially identified in follicular fluid as a naturally occurring gonadal peptide involved in the regulation of the secretion of follicle-stimulating hormone (FSH) by rat anterior pituitary cells. Vale et al., Nature, 321: 776-779 (1986); Ling et al., Nature, 321: 779-782 (1986); DePaolo et al., Proc. Soc. Exp. Biol. Med., 198: 500-512 (1991); Ying, Endocrine Rev., 9: 267-293 (1988).
Subsequent studies of activin revealed other activities, including the effects on follicular granulosa cell differentiation (Sugino et al., Biochem. Biophys. Res. Commun., 153: 281-288 [1988]), spermatogonial proliferation (Mather et al., Endocrinol., 127: 3206-3214 [1990]), erythroid differentiation (EP Publ. No. 210,461 published Feb. 4, 1987; Eto et al., Biochem. Biophys. Res. Commun., 142: 1095-1103 [1987]; Murata et al., Proc. Natl. Acad. Sci. USA, 85: 2434-2438 [1988]; Yu et al., Nature, 330: 765-767 [1987], stimulation of insulin secretion by pancreatic islets (Totsuka et al., Biochem. Biophys. Res. Commun., 156: 335-339 [1988]), enhancement of proliferation of fibroblast (Hedger et al., Mol. Cell Endocrinol., 61: 133-138 [1989]), stimulation of a dose-dependent increase in inositol phosphates in rat parenchymal liver cells, an effect also seen with EGF (Mine et al., Biochem. Biophys. Res. Comm., 186: 205-210 [1992]), modulation of somatotroph functions (Billestrup et al., Mol. Endocrinol., 4:356-362 [1990]), modulation of nerve cell differentiation (Schubert et al., Nature, 344: 868-870 [1990]; Hashimoto et al., Biochem. Biophys. Res. Comm., 173: 193-200 [1990]), and mesoderm induction. Smith et al., Nature, 345: 729-731 (1990); Mitrani et al., Cell, 63: 495-501 (1990).
It has also been found that chronic renal failure serum contains as much activin as normal serum, but the difference between normal serum and the serum of patients with renal failure exists in the context of a specific inhibitor of activin, with the suggestion that activin could be utilized in the therapy of the anemia of such patients. Shiozaki et al., Biochem. Biophys. Res. Commun., 183: 273-279 (1992). While these activities have been demonstrated in vitro, the role of activin in vivo remains poorly understood.
Inhibin and activin are members of a family of growth and differentiation factors. The prototype of this family is TGF-.beta. (Derynck et al., Nature, 316: 701-705 (1985)), which, according to one source, also possesses FSH-releasing activity (Ying et al., Biochem. Biophys. Res. Commun., 135: 950-956 (1986). Other members of the TGF-.beta. family include the Mullerian inhibitory substance, the fly decapentaplegic gene complex, and the product of Xenopus Vg-1 mRNA.
TGF-.beta.1 appears to be a negative regulator of liver growth, and the TGF-.beta. molecule is associated with regression of other epithelial tissues in the embryo (Silberstein and Daniel, Science, 237: 291-293 [1987]) or adult (Kyprianou and Isaacs, supra) and of certain cancers. Kyprianou et al., Cancer Res., 51: 162-166 (1991). Recently, it was reported that cell proliferation and apoptosis are coordinately regulated by TGF-.beta.1 in cultured uterine epithelial cells. Rotello et al., Proc. Natl. Acad. Sci. USA, 88: 3412-3415 (1991). Apoptosis is a physiological cell death wherein the nucleus condenses and the cytoplasm fragments.
Studies in vivo showed that apoptotic hepatocytes in normal and prenoeplastic liver exhibited immunostaining for TGF-.beta.1. Oberhammer et al., Naunyn-Schmiedeberg's Arch. Pharmacol. Suppl., 343: R24 (1991). See also Oberhammer et al., Cancer Res., 51: 2478-2485 (1991). Evidence has now been found that hepatocyte death induced by TGF-.beta.1 in vitro is indeed apoptosis. Oberhammer et al., Proc. Natl. Acad. Sci. USA, 89: 5408-5412 (1992).
A new class of gonadal protein factors, named follistatin or FSH-suppressing protein (FSP), has been isolated from side fractions derived from purifying porcine and bovine ovarian inhibins and activins. Ying, Endoc. Rev., 9: 267-293 (1988); Ling et al., "Isolation and characterization of gonadal polypeptides that regulate the secretion of follicle stimulating hormone," in Hodgen et al., eds., Non-Steroidal Gonadal Factors: Physiological Roles and Possibilities in Contraceptive Development, Jones Institute Press, Virginia, (1988), pp. 30-46. Follistatin was initially characterized by its ability to suppress FSH secretion from the pituitary. The action of follistatin is apparently similar to that of inhibin, but structurally the two proteins are quite different. Ueno et al., Proc. Natl. Acad. Sci. USA, 84: 8282-8286 (1987); Robertson et al., Biochem. Biophys. Res. Commun., 149: 744-749 (1987).
Follistatin is a glycosylated single-chain protein that is found in forms having molecular weights ranging from 31 to 39 kDa. All of these forms have similar amino acid compositions and identical amino-terminal amino acid sequences. The molecular cloning of cDNA with the gene of follistatin revealed two forms, a smaller molecular weight form and a larger form, which are generated by alternative splicing. The smaller form represents a carboxy-terminal truncated form of the larger precursor.
Recent examinations of follistatin gene expression in rat tissues have shown that follistatin mRNA is detected not only in the gonads but also in the kidney, decidual tissue, pancreas, cerebral cortex, pituitary, etc. Shimasaki et al., Mol. Endocrinol., 3: 651-659 (1989); Kaiser et al., Endocrinology, 126: 2768-2770 (1990); Michel et al., Biochem. Biophys. Res. Comm., 173: 401-407 (1990).
It has been found that follistatin is able to neutralize the diverse actions of activin in various systems such as stimulation of FSH secretion by cultured pituitary cells (Kogawa et al., Endocrinology, 128: 1434-1440 [1991]) and induction of mesodermal tissue formation in Xenopus oocytes. Asashima et al., Arch. Dev. Biol., 200: 4-7 (1991). It has been found, in fact, that immunoreactive follistatin is widespread in rat tissues, including hepatic cells, which demonstrated homogeneous immunoreactivity from moderate to strong. Kogawa et al., Endocrinol. Japan, 38: 383-391 (1991). The authors suggest that follistatin is a ubiquitous protein regulating a wide variety of activin actions.
There exists a need for an effective therapy for the prevention of liver damage. This need exists in any patient population in which chronic or acute liver damage has been induced, for example by hepatotoxic compounds, radiation exposure, viral infection, autoimmune disease, elevated in vivo levels of proteins, including liver cell growth inhibitory proteins, hepatotoxic proteins and cytokines, or genetic factors, and where it is desirable to inhibit the progression of such damage. This need further exists in a patient population at risk of developing liver damage, such as in the case of drug overdose, in the case of accidental exposure to infected blood samples, or in a clinical scenario which includes aggressive chemotherapy or radiation therapy.
In many instances, the treatment of serious, life threatening conditions, such as cancer, is severely limited by the hepatotoxicity of the chemotherapeutic agents and/or radiation therapy employed. It would be desirable to be able to expose patients to higher doses of such chemotherapeutics or radiation therapy for an extended period of time without the risk of severe liver damage. There is a related need for an effective liver damage preventative agent which could be included in a clinical protocol potentially inducing liver damage.
It would be particularly desirable to provide means for the prevention of the further progression of liver damage in situations where early intervention is critical. This would be particularly beneficial when known antidotes are no longer effective because of the time elapsed since the exposure to the causative factor of liver damage.
Accordingly, it is an object of the present invention to provide means for the prevention of liver damage in patients at risk of developing liver damage, especially due to hepatotoxic compounds, radiation, or genetic predisposition.
It is another object to provide means for the prevention of the progression of liver damage already occurred.
It is a further object to enable the extended exposure of patients to potentially hepatotoxic treatments and/or to increase the dose of such treatments by preventing the (further) development of liver damage.
It is a still further object of the present invention to provide means for early intervention in patients showing symptoms of a risk of developing liver damage.
It is another object to provide means for preventing the progression of liver damage at a time when antidotes known in the art would no longer be effective.
These and further objects will be apparent to one of ordinary skill in the art.